Abstract
Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis.
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Acknowledgments
We would like to thank Professor Mervyn Bibb (John Innes Centre, Norwich, UK) for providing S. coelicolor M1146, Professor Mark Buttner (John Innes Centre, Norwich, UK) for providing plasmid pIJ10500, and Doctor Weishan Wang (Chinese Academy of Sciences, Beijing, China) for providing promoter pKasO*.
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This work received financial support from the National Natural Science Foundation of China (Grant Nos. 31601701 and 31572070) and General Financial Grant from China Postdoctoral Science Foundation (No. 2016 M600152).
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Haiyan Wang, Xu Cheng, Xiangjing Wang, and Wensheng Xiang designed the research. Material preparation, data collection, and analysis were performed by Xu Cheng, Haiyan Wang, and Yuqing Liu. The first draft of the manuscript was written by Haiyan Wang and Xu Cheng. Shanshan Li and Yanyan Zhang modified the manuscript. All authors read and approved the final manuscript.
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The authors have filed a provisional patent for this work to the China National Intellectual Property Administration (CNIPA). W. X., X.C., H.W., Y.L., and X.W. are inventors on the provisional patent application (CN202010032599.2, filed 13 January 2020).
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Wang, H., Cheng, X., Liu, Y. et al. Improved milbemycin production by engineering two Cytochromes P450 in Streptomyces bingchenggensis. Appl Microbiol Biotechnol 104, 2935–2946 (2020). https://doi.org/10.1007/s00253-020-10410-8
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DOI: https://doi.org/10.1007/s00253-020-10410-8